The objective of this project is to develop a comprehensive set of assays to improve screening and prediction of conditions associated with fragile X syndrome (FXS). FXS, which affects approximately 1/4000 men and 1/6000 women, is caused by an expansion of a cytosine-guanine-guanine (CGG) triplet repeat in the 52 untranslated region (UTR) of the FMR1 gene. The normal FMR1 gene contains up to 40 CGG triplets in the 52 UTR, while the penetrant, full mutation gene is characterized by greater than 200 repeats, hypermethylation of the gene and complete loss of FMR1 protein production. A more modest expansion of the CGG repeats (premutation) has been linked to adult movement, reproductive and cognitive disorders. CGG expansion has also been implicated in autism spectrum disorder (ASD). Generational expansion from premutation to full mutation state can occur when permutation repeats are in the "gray zone" (~30-50 CGG repeats), but interspersed adenine-guanine-guanine (AGG) sequences in the proper locations can confer stability to the gene. The relevance of interspersed AGG sequences in the CGG repeats for predicting expansion to premutation or full mutation alleles has been proposed from population studies of FMR1 gene expansion. It is hypothesized that AGG repeats lead to lower risk of developing Fragile X associated diseases. Therefore, improved testing for fragile X will have important implications for a broad range of individuals of both sexes and all ages at risk of the various conditions associated with this disorder. In these studies we will expand our existing CGG repeat assay to develop a set of assays that will accurately map interspersed AGG sequences that occur within the CGG repeats in the 52 untranslated region of the FMR1 gene. The assays will determine the frequency and positions of these AGG triplets, distinguish between alleles in females, and have the sensitivity to map minor alleles in mosaics, features that are not available on currently available FXS tests. Our goal is a comprehensive diagnostic workflow that is cost-effective and can be routinely used for FXS screening without the need for specialized equipment. The assays will serve as an aid to diagnosis, improve the provision of genetic counseling for FXS and associated disorders, and in the long term, could be used for fetal and newborn screening. Furthermore, population studies of the relevance of AGG interspersion on CGG hyperexpansion will be facilitated with these assays. PUBLIC HEALTH RELEVANCE: Fragile X disease is one of the most common forms of inherited mental retardation, and its genetic cause has been linked to autism, adult reproductive, movement and cognitive disorders. New genetic analysis techniques developed in these studies will provide clinical risk assessment and genetic counseling tools for this devastating group of diseases.